Melanoma, a malignant neoplasm, is derived from cells that are capable of forming melanin, arising most commonly in the skin of any part of the body and in the eye, or rarely, in the mucus membranes of the genitalia, anus, oral cavity, or other sites. It occurs mostly in adults and may originate de novo or from a pigmented nevus or lentigo maligna. In the early phases, the cutaneous form is characterized by a proliferation of cells at the dermal epidermal junction which soon invades adjacent tissues. The cells vary in amount and pigmentation of cytoplasm; the nuclei are relatively large and frequently bizarre in shape, with prominent acidophilic nucleoli; the mitotic figures tend to be numerous. Melanomas frequently metastasize widely; regional lymph nodes, skin, liver, lungs, and brain are likely to be involved.
In January 1985, the Environmental Protection Agency (EPA) predicted that depletion of the Earth's Ozone layer, which guards against ultraviolet (UV) radiation from space, would cause an increase in the number of skin cancer cases worldwide, including melanomas. The EPA estimated an annual increase of two million cases by the year 2050, when the ozone layer is expected to diminish by 10% because of human activities—primarily the release of long-lived Chlorofluorocarbons into the atmosphere (now banned in most developed countries). Public health efforts have focused on encouraging people to use sunscreen, avoid outdoor activities during peak exposure times, perform frequent self-checks of the skin, and visit dermatologists when irregularities are noted. Exposure to higher levels of ultraviolet radiation may also promote cataracts and immune system dysfunction.
UV radiation represents a definitive risk factor for skin cancer, especially when exposure occurs in combination with certain underlying genetic traits, such as red hair and fair skin (1). Pigmentation of the skin results from the synthesis of melanin in the pigment-producing cells, the melanocytes, followed by distribution and transport of pigment granules to neighboring keratinocytes. It is commonly believed that melanin is crucial for absorption of free radicals that have been generated within the cytoplasm by UV and acts as a direct shield from UV and visible light radiation (2, 3).
UV-induced pigmentation (sun tanning) requires induction of α-melanocyte-stimulating hormone (α-MSH) secretion by keratinocytes. α-MSH and other bioactive peptides are cleavage products of Pro-Opiomelanocortin (POMC) (4). The p53 tumor suppressor gene is one of the most frequent targets for genetic alterations in cancer. p53 is a transcriptional regulator of the POMC gene, which translates to proteins that cause the melanocytes to produce melanin, which wards off skin cancer by absorbing UV radiation. Direct mutational inactivation of p53 is observed in close to half of all human tumors (5).
Malignant melanoma is a skin cancer which is, by far, one of the hardest cancers to treat today. Dacarbazine (DTIC) is the only single agent used to treat metastatic malignant melanoma. However, in the clinical setting the Complete Response (CR) rate for Dacarbazine is below 10% and hence is an unmet medical need and there exists a need for better agents. In addition. Dacarbazine is also indicated for Hodgkin's lymphoma as a secondary line therapy when used in combination with other effective drugs. Chemically. DTIC is 5-(3,3-dimethyl-1-trizeno)-imidazole-4-carboxamide with the following structural formula:

Dacarbazine, however, requires bioactivation in vivo by the liver. One of the methyl groups of the dimethyltriazeno functionality is activated by liver microsomal enzymes and, in particular, by the Cytochrome P450, to oxidation, resulting in a hydroxymethyl group. Thus, the oxidative mono-demethylation of the dimethyltriazeno functionality affords monomethyltriazene. The monomethyltriazene metabolite, 3-methyl-(triazen-1-yl)-imidazole-4-carboxamide (MTIC) is further hydrolyzed to 5-amino-imidazole-4-carboxamide (AIC), which is known to be an intermediate in purine and nucleic acid biosynthesis and to methylhydrazine, which is believed to be the active alkylating species. The Cytochrome P450 enzymes play only a minor role in the metabolism of MTIC.
Temozolomide is also a similar imidazotetrazine alkylator that methylates DNA at nucleophilic site. Temozolomide is orally bioavailable, more lipophilic, and spontaneously converted to MTIC, and also seems to generate less nausea (6). The O6-methylguanine adduct causes a mismatch during DNA replication and the addition of a thymidine, instead of cytosine, to the newly formed DNA strand (7). Because of the excellent CNS biodistribution, temozolomide has been useful as a radiosensitizer in both primary brain tumors and CNS metastases (8-11). The pharmacokinetics of temozolomide has been studied in children, and clearance is related to body surface area (12). Temozolomide improves quality of life when used with radiation in patients with brain metastases. Unlike Dacarbazine, Temozolomide has activity against sarcoma (13-15). Thus, it may be useful in sarcoma radiosensitization for primary control as well as for the treatment of metastases. Temozolomide is a radiosensitizer that is well tolerated and has modest side effects. The combination of Temozolomide and Irinotecan is more than additive against some cancers (16). The authors report that their experience confirms a high response rate in relapsed Ewing's sarcoma and DSRCT that is possibly even higher than that reported in the literature (17-19). The Temozolomide plus Irinotecan combination is less immune suppressive than standard cyclophosphamide-containing regimens (20). This might be especially important in Ewing's sarcoma since these authors and others have shown that lymphocyte recovery (i.e., absolute lymphocyte count >500 on day 15 after the first cycle of chemotherapy) is associated with significantly higher survival in Ewing's sarcoma (21, 22). Temozolomide or Dacarbazine has also been combined with other drugs including Gemcitabine and Doxorubicin liposomes (23, 24). The disappearance of DTIC from the plasma is biphasic with an initial half life of 19 minutes and a terminal half life of five hours. In a patient with renal and hepatic dysfunctions, the half lives were lengthened to 55 minutes and 7.2 hours, respectively. The average cumulative excretion of unchanged DTIC in the urine is 40% of the injected dose in six hours. DTIC is subject to renal tubular secretion rather than Glomerular Filtration. At therapeutic concentrations, DTIC is not appreciably bound to human plasma protein.
In humans, DTIC is extensively degraded. Besides unchanged DTIC, AIC is a major metabolite of DTIC excreted in the urine. Although the exact mechanism of action of DTIC is not known, three hypotheses have been offered:    1. Inhibition of DNA synthesis by acting as a purine analog    2. Acting as an alkylating agent    3. Interaction with SH groups    Thus, the biochemical mechanism of action of the resulting MTIC reactive species whose cytotoxicity involved generation of methyl carbonium ion in vivo is thought to be primarily due to alkylation of DNA. Alkylation (methylation) occurs mainly at the O6 and N7 positions of guanine.
Alternatively, DTIC, prior to its metabolism to the monomethyltriazene, is oxidized initially to monohydroxymethyl and finally to an aldehyde. The monomethyltriazene, in its aldehyde form prior to oxidative monodemethylation, is cyclized to the cyclic compound (as shown in Scheme 1) which interferes with the double helix DNA structure and blocks replication of the cancer cells. And finally, the secondary metabolite, AIC, is inactive.
The imidazole ring system of the Dacarbazine is hydrophilic in nature. Therefore, there is a need in the art for possibly effective binding to the melanin such that the cytotoxic functionality of the molecule is one hundred percent effective. Thus, the present inventors have aimed to provide novel compounds with increased lipophilicity thereby providing more target specificity. Thus, Thiophene, which has a five-membered heterocyclic ring system, is lipophilic in nature and may have effective binding by increased avidity to the melanin, as a result, one would be able to get the same therapeutic effectiveness at a significantly lower dose, hence minimizing the toxicity. This would in turn afford high specificity with a larger window of the Therapeutic Index (TI). In general, for the treatment of cancer patients, a larger therapeutic index is preferred. This is because, one would like to start the therapeutic regimen with a very high Maximum Tolerated Dose (MTD) such that the cancer cells would be hit hard in the first chemotherapy itself. Otherwise, the surviving cancer cells would repair the DNA damage and subsequently metastasize to the other organs. In addition, the cancer cells that survived from the first treatment would become resistant to the second chemotherapy again, if needed. And besides, due to weakness of the immune system from the first chemotherapy, a suboptimal dose would be given in the second treatment that would contribute to toxicity.
As shown in scheme-1, unlike DTIC, better interaction of the thiophene ring system with the SH groups on the surface of the tumor antigen results in increased efficacy. This is because of sulfur (S) being larger atom and hence a five membered heterocyclic aromatic thiophene ring system resemble a phenyl ring in space, would contribute it's loan pair of electrons to the rest of the ring for better interaction with sulfhydryls at the tumor site. In addition, due to it's electronic configuration, the heterocyclic aromatic thiophene ring system may be superior over DTIC by way of inhibition of DNA synthesis by acting as a purine analog as well as acting as an alkylating agent. Also, unlike DTIC, while Amino Imidazole Carboxamide (AIC) is inactive, the corresponding Amino Thiophene Carboxamide (ATC) would very well be active in vivo via de-localization of electrons from the ring sulfur for increased efficacy. Thus, the novel triazeno thiophene analogs have several additional advantages inherently built in within the structure over dacarbazine for increased activity.
In addition to its biochemical mechanism of action, recently there are several reports in the literature for significantly increasing the efficacy of Dacarbazine by using it in combination with other chemotherapeutic agents (25, 26). Likewise, in a pre-clinical setting, nanoemulsion preparations of Dacarbazine in a xenograft mouse melanoma model has been used to significantly increase it's efficacy (27). Similarly, a number of innovative therapeutic strategies have been pursued in order to improve the outcomes, including immune therapy, tyrosine kinase inhibitors and angiogenesis inhibitors (28). The literature reports treatment for metastatic melanoma using Dacarbazine in combination with interferons is poor (29). As described above, currently, dacarbazine and temozolamide have been extensively used chemotherapeutic agents for treating metastatic malignant melanoma. However, the success rate is low and the side effects are high. Hence, there exists an unmet medical need for the development of effective agents and approaches for treatment of metastatic malignant melanoma remains an immense challenge.